The Need For Directing Therapeutic Agents to Selected Cells.
Therapeutic agents are agents administered with the intent of changing, in a beneficial manner, some physiological function of the recipient. Such agents can include drugs, proteins, hormones, enzymes, nucleic acids, peptides, steroids, growth factors, modulators of enzyme activity, modulators of receptor activity and vitamins. This invention involves directing therapeutic agents to selected cells (which is also called targeting), thereby increasing the concentration of therapeutic agent in some cells where the agent produces a beneficial effect and decreasing its concentration in cells where it produces a toxic effect. By directing the therapeutic agent toward certain cells where drug efficacy is to be obtained, and away from other cells where drug toxicity is obtained, the safety and efficacy of an agent can be improved.
In contrast to therapeutic agents, diagnostic contrast-type agents are administered with the intent of illuminating some physiological function, while leaving other physiological functions unaffected. These diagnostic agents include radioactive isotopes for scintigraphy, electron dense labels for X-ray or computer tomography, and magnetic labels for magnetic resonance imaging.
RES Based Targeting
One strategy of targeting therapeutic agents involves directing such agents to the phagocytic cells, called macrophages, which are found in high numbers in a series of organs referred to as the reticuloendothelial system (RES). Organs of the RES include the liver, spleen and bone marrow. Phagocytosis is a process whereby a wide variety of materials, including colloids, particles, liposomes and microspheres are non-specifically removed from the blood. For example, Imferon, a dextran coated colloidal ferric oxyhydroxide used for the treatment of anemia, is slowly cleared from the blood by the phagocytic activity of the macrophages of the RES. (Henderson et al., 34 Blood (1969) pp. 357-375.). Liposome encapsulated drugs have also been used to treat such diseases as Leishmania (O'Mullane et al "Biopharmaceutics of Microparticulate Drug Carriers," Ann. N.Y. Acad. Sci. (1987) 507:120-140) Microspheres have also been employed to deliver agents to the RES, often to stimulate the immune function of macrophages (Kanke et. al. "Interaction of Microspheres with blood constituents, III. Macrophage phagocytosis of various types of polymeric drug carriers," 42 J. Parenteral Science and Technology (1988) pp. 157-165). However, directing therapeutic agents to macrophages is of little use in many diseases that do not involve macrophages or macrophage function.
RME Based Targeting
A second strategy for targeting therapeutic agents to macrophages involves attaching agents to molecules (termed carriers) that are withdrawn from the vascular compartment by receptor mediated endocytosis (RME). RME is a process whereby molecules in the extracellular space bind to specific receptors on the cell surface and are internalized. The uptake of substances by RME is a feature of normal, healthy cells. RME transport systems can be found on normal macrophages, hepatocytes, fibroblasts and reticulocytes. RME enables cells to remove a variety of macromolecules from plasma, such as low density lipoproteins, transferrin and insulin. See Table 1 of Wileman et al., 232 Biochem. J. (1985) pp. 1-14 for a list of cells performing RME, which also contains a general review of RME. See also Table I of Menz, E. T., PCT WO 90/01295, filed Aug. 3, 1989, both incorporated herein by reference. By attaching therapeutic agents to carriers undergoing RME, therapeutic agents can be directed to cells which do not perform phagocytosis, e.g., hepatocytes of the liver. Targeting therapeutic agents based on RME requires the attachment of therapeutic agents to satisfactory carrier molecules, which then alter the biodistribution of the therapeutic agent.
Diagnostic agents have also been attached to carriers that that undergo uptake by RME, for example, carriers that interact with asialoglycoprotein receptor such as, radioisotope preparations of neoglycoalbumin-.sup.99 Tc have shown high liver specificity in animal studies (Vera et al, J. Nucl. Med. 26:1157-1167 1985). In another example, Groman et al, (U.S. Pat. No. 5,284,646, incorporated herein by reference) conjugated superparamagnetic metal oxides to glycoproteins for use as an in vivo contrast agent in magnetic resonance imaging.
Carriers For RME Targeting
One type of molecule widely employed as carriers for delivering therapeutic agents based on RME are glycoproteins. A glycoprotein molecule consists of a protein backbone that is associated with multiple oligosaccharide side chains, which often consist of between 2 and 20 monosaccharides covalently linked to the protein backbone by either an N-linkage or an O-linkage (Stryer L, Biochemistry, 3d Ed., N.Y.: W. N. Freeman Co., pp. 343-344 (1988)). For example, a receptor known as the asialoglycoprotein receptor on hepatocytes recognizes glycoproteins possessing galactose residues and interalizes them. Those glycoproteins that have a sialic acid attached to a penultimate galactose on the associated oligosaccharides lack an affinity for the receptor, but can be converted to receptor binding molecules by removal of the terminal sialic acid to expose the galactose. For example, fetuin can be converted to asialofetuin by removal of the terminal sialic acid groups. Recognition by the asialoglycoprotein receptor, which performs RME, is dependent on the number and clustering arrangement of the galactosyl linkages on the oligosaccharide. Similarly, the mannose receptor on macrophages recognizes glycoproteins possessing mannose residues and internalizes them by RME.
An alternative to glycoprotein carriers are the so-called neoglycoproteins, which are synthesized when multiple mono- or disaccharides are covalently attached to protein molecules. An example of a neoglycoprotein is lactosylated bovine serum albumin, which binds to the asialoglycoprotein receptor on hepatocytes.
Table 1 provides selected examples of the receptors, cells, therapeutic agents and carriers involved with RME based targeting. For further reviews see Ranade, J. Clin. Pharmacol. 29:685-694 (1989); and Bodmer et al., Methods of Enzymology vol. 112., p. 298, Academic Press (1985). For recent reviews, see Meijer, Antiviral Research, 18:215-258 (1992); Meijer, Trends in Drug Research, vol 13, 303-332; Meijer, Pharm. Res. 6(2):105-118 (1989).
TABLE 1 __________________________________________________________________________ RME Based Targeting: Receptors, Cells, Therapeutic Agents and Carriers Receptor/Cell Therapeutic Agent/Carrier Reference __________________________________________________________________________ Galactose or Asialoglycoprotein/ ara AMP/lactosylates human serum Fiume et al., Lancet 2:13-15 Hepatocytes albumin (1988) Galactose or Asialoglycoprotein/ acetylcyteine/asialofetuin Wu and Wu, Hepatology 5 709-713 Hepatocytes (1985) Galactose or Asialoglycoprotein/ folinic acid/asialofetuin Wu and Wu, Proc. Natl Acad. Sci. Hepatocytes 80:3078-3080 (1983) Galactose or Asialoglycoprotein/ DNA/asialoorosomucoid Wu and Wu, J. Biol. Chem. Hepatocytes 263:14621-14624 (1988) Mannose/T4 lymphocytes AZT/mannosylated albumin Molema et al, Biochem. Pharm. 40 2603-2610 (1990) Mannose/macrophage muramyl dipeptide/mannosylated albumin Roche et al., Res. Virol. 141, 243-249. __________________________________________________________________________
Features of RME
Uptake by RME exhibits three properties characteristic of ligand-receptor interactions generally: structural specificity, saturability and competition.
Structural specificity is observed when a receptor can distinguish between closely related structures and only molecules with structures meeting the binding requirements of the receptor binding site are internalized. Often the receptors involved in RME are discovered by their ability to internalize or clear glycoproteins from circulation.
Saturability is observed when the rate of an agent internalized via RME decreases with increasing concentrations of that agent. This results because, at high concentrations, the receptor approaches full occupancy or becomes saturated with ligand.
Competition is observed when the removal from the blood (clearance) of an molecule can be reduced by the presence of a second molecule bearing a structural resemblance to the first agent. The second molecule competes for receptor binding sites and decreases the rate of internalization of the first agent. Whereas saturability occurs when high concentrations of a single ligand compete for a limited number of receptor sites, competition results when chemically different ligands bind to a limited number of receptor sites. Competition is used to distinguish RME type polysaccharides of the invention from other types of polysaccharides (see Table 2).
Problems with Glycoproteins and Neoglycoproteins as Carriers for Therapeutic Agents
In spite of the many cases where glycoproteins and neoglyproteins have been used as carriers of therapeutic agents, these carriers are subject to several problems, some of which have been discussed in the literature.
1. Glycoproteins and neoglycoproteins undergoing RME are not naturally occuring materials. Modification of the glycoprotein, such as the removal of a terminal sialic acid to expose the galactose residue, is required for the glycoprotein to interact with the asialoglycoprotein receptor. For example, fetuin must be desialylated to produce a carrier that can interact with the receptor. Similarly, neoglycoproteins are synthesized by attaching multiple lactose residues to albumin.
2. Glycoproteins derived from bovine sources can be immunogenic in humans. Neoglycoproteins have been reported to be immunogenic in animals (Fiume L., Busi C., Preti P., Spinosa G. Cancer Drug Delivery, 1987, 4:145-150).
3. Neoglycoproteins and glycoproteins generally will not tolerate organic solvents during conjugate synthesis. Organic solvents are employed in the examples of the invention.
4. With the synthesis of neoglycoproteins, the positively charged amine groups of proteins are utilized (and neutralized), for the attachment of mono or disaccharide. As a result, the neoglycoprotein is often strongly negatively charged. Such strong negative charge facilitates uptake by so-called scavenger receptors, and decreases the amount of therapeutic agent delivered to cells by RME (Kanke M., Geissler R. G., Powell D., Kaplan A., DeLuca P. J. Parenteral Science and Technology, 1988, 42:157-165).
For the foregoing reasons, there is a need for new approaches to direct therapeutic agents to selected cells, a need which could be met if improved carriers for delivering therapeutic agents to cells performing RME could be found. Such carriers should have a high affinity for the receptor, and maintain that affinity after complex formation and attachment of the therapeutic agent. In addition, the desired carrier should be naturally occurring, be readily available in pure form, and must be nontoxic.